Recombinant platelet collagen receptor glycoprotein VI and its pharmaceutical use

ABSTRACT

The invention relates to Glycoprotein VI (GPVI), its isolation, purification, and methods for recombinant production. Especially, the invention relates to the use of GPVI, preferably recombinant GPVI, in the treatment of disorders and pathological events correlated directly or indirectly to blood coagulation disorders such as thrombotic and cardiovascular diseases. The extracellular recombinant protein can also be used for establishing screening assays to find potential inhibitors of the membrane bound GPVI in order to inhibit binding of thrombocytes and platelets, respectively, to collagen. Changes in GPVI can be used to monitor platelet age and exposure to thrombotic and cardiovascular diseases.

This application is a divisional application of U.S. Ser. No.09/959,802, filed Nov. 7, 2001, now U.S. Pat. No. 7,928,066, which is aU.S. National Stage application under §371 of PCT/EP00/03683, filed Apr.25, 2000, the entirety of disclosures in which are incorporated byreference herein.

The invention relates to Glycoprotein VI (GPVI), its isolation,purification, and methods for recombinant production. Especially, theinvention relates to the use of GPVI, preferably recombinant GPVI, inthe treatment of disorders and pathological events correlated directlyor indirectly to blood coagulation disorders such as thrombotic andcardiovascular diseases. The extracellular recombinant protein can alsobe used for establishing screening assays to find potential inhibitorsof the membrane bound GPVI in order to inhibit interaction of plateletsand collagen. GPVI on the platelet surface is modified during theplatelet lifetime in vivo and can therefore be used as a marker of theplatelet age profile.

Glycoprotein VI is a 62/65 kDa (non-reduced/reduced respectively)platelet membrane glycoprotein which forms a complex together with theFcγ common subunit. The GPVI subunit contains the collagen binding siteand the Fcγ subunit is responsible for signalling. The complex forms oneof the major collagen receptors on the platelet surface, critical forplatelet activation in response to collagen. The recognition sequence oncollagen consists of (GlyProHyp)_(n) sequences. Patients are known fromJapan who have a genetic deficiency of GPVI. They have mild bleedingproblems and their platelets respond only weakly to collagen, presumablyvia other receptors. A great deal has been learned about the signallingcascades originating at GPVI which strongly resemble those from immunereceptors including T-cell receptors, B-cell receptors and naturalkiller cell receptors. These cascades involve src family tyrosinekinases such as Fyn and Lyn as well as p72^(SYK) and many other tyrosinekinases and phosphatases and adaptor proteins such as LAT. A main targetof these cascades is activation of phospholipase Cγ2 which splitsphospholipids to give the second messengers diacylglycerol and IP₃. GPVIis thought to be involved in activation of the platelet integrin α2β1which has a major role in platelet adhesion to damaged vessel wall. Micewith the Fcγ subunit “knocked-out” have platelets which still showresponses to collagen implying that the resting state of α2β1 may alsobe regulated by the GPVI/Fcγ complex.

The platelet collagen receptor GPVI is closely related to the naturalkiller activatory receptors of the p58KAR family as well as to FcαR.

The adhesion and activation of resting, circulating platelets at a siteof vascular injury is the first step in a process leading to theformation of a thrombus which is converted into a haemostatic plug.Collagen is one of the major components of the vessel wall responsiblefor platelet activation. Many types of collagen exist and seven of theseare found in the subendothelial layers. Several different receptors forcollagen have been identified on platelets but the major ones are nowconsidered to be the integrin α₂β₁ and the non-integrin GPVI. Althoughα₂β₁ is well characterised and both subunits were cloned and sequencedseveral years ago, the structure of GPVI has remained elusive althoughseveral features have been identified. It was determined about twentyyears ago that GPVI is a major platelet glycoprotein with a molecularmass in the 60-65 kDa range and an acid pI. Its role as a putativecollagen receptor was established following the identification of apatient in Japan with a mild bleeding disorder whose platelets showed aspecific defect of response to collagen and lacked this receptor. Thispatient had also developed autoantibodies to the deficient receptor andthese were used to characterise the molecule further. More recently itwas established that GPVI is associated non-covalently with the commonFcγ subunit which acts as the signalling part of the complex. It wasalso demonstrated that the recognition sequence on collagen for GPVI isa repeated Gly-Pro-Hyp triplet within the collagen triple helicalstructure and that synthetic peptides based on this structure could beused as specific GPVI directed agonists. The GPVI/Fcγ complex was shownto signal to the platelet interior by an immune receptor-like mechanism,involving activation of p72^(SYK) and leading by a cascade ofkinase/phosphatase/adaptor protein interactions to activation of PLCγ2and hence to release of granules and platelet aggregation. A furtherstep in characterisation of this molecule was the demonstration that thesnake C-type lectin, convulxin, from the Tropical Rattlesnake, Crotalusdurissus terrificus was able to activate platelets by clustering GPVIthrough a multimeric interaction. Convuxin was shown to bindspecifically to GPVI providing a method for purification of thisreceptor in conjunction with established approaches. Thus, it is clearfrom the prior art that GPVI seems to be a very interesting compound inmany therapeutical fields above all concerning with applications whichare related, directly or indirectly, to blood coagulation events whichdepend on collagen—platelet interaction. It was, therefore, the goal ofthe present invention is to provide GPVI in a recombinant form and toshow its efficiency as direct therapeutical target or as tool forscreening of short compounds, especially chemically synthesized orsynthesizable compounds having the capability to inhibit or block thenatural platelet-collagen interaction.

The invention relates also to portions or fragments of the GPVI proteinwhich have maintained their biological activity which is the binding tocollagen.

The invention was successful in purifying adequate amounts of GPVI forpreliminary characterisation and for peptide sequencing. The sequenceswere used to design primers for PCR to identify a positive sequence in aDNA library. This DNA sequence was then used as a probe to isolate analmost complete cDNA sequence from the library and missing 5′-sequencewas obtained using a RACE method from a platelet cDNA library.

The invention was also successful in showing the use of recombinant GPVIas therapeutically applicable compound which is capable, whenadministered in a patient with e.g. damaged blood vessels, to bind tocollagen, thus preventing platelets bearing membrane-bound GPVI frombinding to said collagen. The recombinant soluble extracellular domainof GPVI contains the collagen binding site and can prevent plateletactivation by collagen. It could therefore be applicable to treatment ofdisease conditions involving increased platelet activation withcollagen, such as atherosclerotic plaque rupture, in diseases such asunstable angina or, during surgical treatment such as PercutaneousTransluminal Coronary Angioplasty (PTCA), where arteries are reopened byinflation of a balloon catheter causing considerable damage to thevessel wall and much platelet activation and often resulting inreclosure of the vessel later. The advantage of recombinant GPVIfragments compared to present treatment methods is that they act at anearlier stage by preventing or reducing platelet activation rather thanby suppressing events after platelet activation, such as aggregation byGPIIb-IIIa antagonists. Thus, smaller amounts of platelet granulecontents are released including growth factors and chemokines which areinvolved not only in wound repair but in the remodelling of the vesselwall by smooth muscle migration and in attraction of phagocytic cellssuch as monocytes known to contribute to atherosclerosis. Fab fragmentof humanised mouse monoclonal antibodies against GPVI could be used withsimilar effect to block GPVI on the platelet surface with similarapplications as above.

Recombinant GPVI according to this invention can also be used in abinding assay to collagen to screen for small molecules (in combinatorylibraries for example) capable of inhibiting this interaction and whichcan be used to develop therapeutic compounds which are inhibitors of thecollagen-platelet interaction. By suitable derivatisation thesecompounds are made orally available. Again the main objective is toprepare compounds reducing GPVI-collagen interactions and hence plateletactivation in situations where platelets come into contact withcollagen. The screening technology as such used in this invention iswell established in the prior art. By such screening assays theinvention enables finding and developing new targets which can inhibitthe natural membrane-bound GPVI on the platelet surface as a collagenantagonist. Such targets which may be small chemical molecules may thenbe the basis for further inventions.

Another major application of GPVI and reagents that recognize specificdomains of GPVI is as markers of platelet age and functionality. Youngplatelets are generally thought to be more active and functional thanolder ones. Young platelets bind to and are activated by the snake venomC-type lectin convulxin, which is specific for GPVI, and as they ageboth the binding and degree of activation decrease. This can be due toeither proteolytic or conformational changes in GPVI or its associationwith Fcγ due to platelet activation or damage in the circulation. Thiscan be a useful parameter to measure the age and function profile ofplatelets in patients as well as in normal persons during medicalcontrols. The platelet age profile changes in many diseases affectingthe bone marrow or the immune system and could be an importantdiagnostic criterion if better methods for its determination wereavailable. For example, patients with diseases involving increasedplatelet turnover will show more young platelets whereas patients onchemotherapy or radiation treatment will show a steadily agingpopulation. Thus, such an age profile can be used for a precisemonitoring of treatment. In a normal healthy population very little isknown about the age to profile distribution and its role as a predictorof changes in health. It is not known whether the changes in GPVI aredue to the partial involvement of platelets in haemostatic events andwhether changes might be more pronounced in patients with extensivecardiovascular disease. At present thiazole orange is used to detectyoung reticulated platelets containing mRNA. This mRNA soon decays,restricting the method to only the youngest platelets. Reagents whichcould be used in such an assay would include GPVI-specific snake venomproteins such as convulxin, or monoclonal or polyclonal antibodiesrecognising the N-terminal region of GPVI or monoclonal antibodiesrecognising new sites or conformations exposed by proteolysis of theN-terminal domain or specific conformations present either in the intactmolecule and not in the aged one or vice versa or small chemicalentities selected to recognise specifically intact GPVI or its modifiedform. These reagents would be labelled with a fluorescent marker, ortogether with a fluorescent labelled second antibody or affinity reagentand used in flow cytometry to measure the platelet binding profile. At alater stage alternative, less labour intensive measuring techniquesbased on automated measuring of platelet profiles could be adopted.Using cell sorting methods with flow cytometry or magnetic beads itshould be possible to isolate young and old platelets to examine thefactors involved in removal of old platelets from the circulation.Reagents recognizing specific forms of GPVI would be a key to suchstudies.

Therefore, it is an object of the present invention to provide a DNAcoding for Glycoprotein VI or biological active fragments thereof,especially the sequence of FIG. 2.

It is a further object of this invention to provide a DNA coding forGlycoprotein VI comprising the amino acid sequences of FIGS. 1 a and 1b.

It is another object of this invention to provide a pharmaceuticalcomposition comprising recombinant GPVI together with a pharmaceuticallyacceptable diluent, carrier or excipient, and its use for themanufacture of a medicament in the therapeutical field of thrombotic andcardiovascular events and disorders related to platelet-collageninteractions.

Another object of the invention is the use of recombinant GPVI in ascreening tool for detecting specific inhibitors of platelet-collageninteractions.

Another object of the invention is the use of GPVI as a marker forplatelet age and exposure to cardiovascular diseases.

Possible medical indications and applications, respectively, are, forexample, unstable angina pectoris, PTCA, use of stents in this field,operations on coronary vessels, general operations on blood vessels,operations which may damage larger blood vessels such as hip jointoperations. Moreover, all indications are included which relate tothromboembolic events caused by disorders of the interaction between thevessel wall and the coagulation system with a high risk of formation ofthrombi and blocking of vessels.

As indicated above, the GPVI protein and fragments thereof according tothe present invention are suitable as pharmaceutically effectivecompounds in pharmaceutical compositions and combinations.

The pharmaceutical formulations according to the invention optionallymay comprise additional active ingredients like anti-coagulants such ashirudin or heparin or thrombolytic agents such as plasminogen activatoror hementin or antagonists to other platelet receptors such asGPIIb-IIIa antagonists like abciximab or eptifibatide or ADP-receptorantagonists such as clopidogrel.

The novel protein, and its biological active fragments respectively,according to the invention may form pharmaceutically acceptable saltswith any non-toxic, organic or inorganic acid. Inorganic acids are, forexample, hydrochloric, hydrobromic, sulphuric or phosphoric acid andacid metal salts such as sodium monohydrogen orthophosphate andpotassium hydrogen sulfate. Examples for organic acids are the mono, diand tri carboxylic acids such as acetic, glycolic, lactic, pyruvic,malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic,maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic,salicylic and sulfonic acids such as methane sulfonic acid. Salts of thecarboxy terminal amino acid moiety include the non-toxic carboxylic acidsalts formed with any suitable inorganic or organic bases. These saltsinclude, for example, alkali is metals such as sodium and potassium,alkaline earth metals such as calcium and magnesium, light metals ofGroup IIIA including aluminium, and organic primary, secondary andtertiary amines such as trialkylamines, including triethylamine,procaine, dibenzylamine, 1-ethenamine, N,N′-dibenzylethylene-diamine,dihydroabietylamine and N-alkylpiperidine.

As used herein, the term “pharmaceutically acceptable carrier” means aninert, non toxic solid or liquid filler, diluent or encapsulatingmaterial, not reacting adversely with the active compound or with thepatient. Suitable, preferrably liquid carriers are well known in the artsuch as sterile water, saline, aqueous dextrose, sugar solutions,ethanol, glycols and oils, including those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil andmineral oil.

The formulations according to the invention may be administered as unitdoses containing conventional non-toxic pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles which are typical forparenteral administration.

The term “parenteral” includes herein subcutaneous, intravenous,intra-articular and intratracheal injection and infusion techniques.Also other administrations such as oral administration and topicalapplication are suitable. Parenteral compositions and combinations aremost preferably adminstered intravenously either in a bolus form or as aconstant fusion according to known procedures. Tablets and capsules fororal administration contain conventional excipients such as bindingagents, fillers, diluents, tableting agents, lubricants, disintegrants,and wetting agents. The tablets may be coated according to methods wellknown in the art.

Oral liquid preparations may be in the form of aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or anothersuitable vehicle before use. Such liquid preparations may containconventional additives like suspending agents, emulsifying agents,non-aqueous vehicles and preservatives.

Topical applications may be in the form of aqueous or oily suspensions,solutions, emulsions, jellies or preferably emulsion ointments.

Unit doses according to the invention may contain daily required amountsof the protein according to the invention, or sub-multiples thereof tomake up the desired dose. The optimum therapeutically acceptable dosageand dose rate for a given patient (mammals, including humans) depends ona variety of factors, such as the activity of the specific activematerial employed, the age, body weight, general health, sex, diet, timeand route of administration, rate of clearance. the object of thetreatment, i.e., therapy or prophylaxis and the nature of the thromboticdisease to be treated, antiplatelet or anticoagulant activity.

Therefore, in compositions and combinations useful as anticoagulants ina treated patient (in vivo) a pharmaceutical effective daily dose of thepeptides of this invention is between about 0.01 and 100 mg/kg bodyweight, preferably between 0.1 and 10 mg/kg body weight. According tothe application form one single dose may contain between 0.5 and 10 mgof the collagen inhibitor to achieve an anticoagulant effect inextracorporeal blood a pharmaceutically effective amount of theinventive peptides is between 0.2 and 150 mg/l, preferably between 1 mgand 20 mg/l extracorporeal blood.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1: Protein sequence of GPVI (one-letter-code)

-   -   1a: Leader sequence (SEQ ID NO: 2)    -   1b: Mature protein (SEQ ID NO: 3)

-   Open reading frame: 339 amino acids

-   Asterisk: Glycosylation site

-   Double underline: Transmembrane domain

-   Underline: Sequenced peptides

FIG. 2: GPVI nucleotide sequence (SEQ ID NO: 1) covering open readingframe of 1017 bp plus 3′ and 5′ regions total 1249 bp

DETAILED DESCRIPTION OF THE INVENTION

Two sequences of 7 amino acids showing the least degeneracy in thegenetic code were chosen for the synthesis of DNA primers in order toamplify part of the of GPVI cDNA by PCR. As the location of bothpeptides in the protein were totally unknown, for each of them, twodegenerate primers, one sense and one antisense were prepared. Theseprimers were used to amplify a human bone-marrow library. Thecombination of the sense 5′ TYA THC CNG CNA TGA ARMG 3′ (SEQ ID NO: 4)primer coding for the sequence PAMKRSL (SEQ ID NO: 5) with the antisense5′ TTR TAN ARN GCR AAY TGR TC 3′ (SEQ ID NO: 6) one corresponding toDQFALYK (SEQ ID NO: 7) amplified a DNA fragment of 221 bp. In additionto the selected peptides, the amplified DNA coded for the LysC/AspNpeptide DQLELVATGVFAKPSLSAQPGPAVSS (SEQ ID NO: 11), clearly linking thesequence to the cDNA for GPVI.

Screening 600.000 pfu from a bone marrow library with this 221 bp DNAfragment produced 4 positive pfu. Three had inserts of 1350 bp whethercut by the restriction enzymes Sal I or EcoR I and belonged to the IgGsuperfamily. The fourth one had an 4.6 kb insert by Sal I digestion andgave two fragments of 2300 bp and 1300 bp respectively when treated byEcoRI. Its DNA encoded the sequence for the 10 peptides derived fromamino acid sequencing of GPVI but stopped short of the amino terminal.No starting methionine or leader sequence could be found but more than2000 bp of previously sequenced non-reading frame DNA terminating in anAlu sequence were present. The 5′ end RACE experiment was completed onplatelet poly A RNA with primers located in a part of the GPVI sequencewhich had been corroborated by that of the peptides. A fragment of 348bp including 278 bp on the sequence of the fourth clone and 70 bp newfrom by 1987 corresponding to 14 amino acids including the firstmethionine were found before falling back on the established GPVIsequence. Thus, a cDNA containing a total of 1249 bp, a 25 bp 5′sequence upstream of the start codon, an open reading frame of 1017 bpcoding for a protein, including leader sequence, with 339 amino acids,and a 3′ region of 207 bp including the stop codon could be sequenced.

A cDNA coding for platelet GPVI was cloned and sequenced from a humanbone marrow cDNA library using RACE with platelet mRNA to supply missing5′ sequence. The open reading frame of 1017 bp encodes 339 amino acidsand a untranslated 3′ region. Hydrophobicity analysis of the amino acidsequence revealed the presence of two putative transmembrane domains, aputative 20 amino acid signal sequence, and a 19 amino acid domainbetween residues 247 and 265 of the mature protein. The sequence and itsamino acid translation are shown in FIG. 2 and FIG. 1. A comparison withthe amino acid sequence of the most similar molecules found in a searchof GenBank reveals clearly that it belongs to the immunoglobulinsuperfamily and the extracellular domain contains two Ig C2-domain loopsformed by two disulphide bridges. It is a membrane crossing proteinclass one molecule with the N-terminus at the exterior and traverses themembrane once. The most closely related molecules belong to the naturalkiller receptor class which contains both inhibitory and activatorytypes. GPVI clearly belongs to the activatory subclass not only throughits function but also because unlike the inhibitory class it does notcontain ITIM sequences in its cytoplasmic domain. Neither does itcontain any tyrosine residues which might be involved inphosphorylation. There are some threonine and serine residues in thisdomain but they do not match any criteria for kinase consensussequences. Like the activatory class of NK receptors, GPVI contains anarginine residue as the third amino acid of the membrane crossing domainwhich is involved in the complex formation with the Fcγ subunit. Thecytoplasmic domain contains 51 amino acids, showing only a minorsimilarity (in the region just below the membrane) to the cytoplasmicdomains of other members of this family. This suggests that this domainin GPVI may associate with different types of cytoplasmic molecule thanthe other family members. GPVI contains only a single putativeN-glycosylation site at Asn69. The domain just above the membrane afterthe beta sheets of the Ig domains finish, however, is rich in theonineand serine residues which could provide O-glycosylation sites such asare found in GPIbα and GPV. The main function of this O-glycosylationseems to be to present the receptor structures well-extended from theplatelet surface to facilitate the interactions with their bulkyligands. Since GPVI was earlier established as a sialoglycoprotein, thedifference in molecular mass between the theoretical amino acid mass (37kDa) and the mass determined by gel electrophoresis (65 kDa reduced)must be due to this glycosylation.

The structure of natural killer receptors of the two domain type hasbeen established by X-ray crystallographic studies and the twoIg-domains were shown to form an acute angle with the receptor site forthe peptide-carrying HLA antigens lying on the outside of the elbow. Adirect comparison of the structure of the HLA peptide binding site withthat of collagen immediately suggests why these receptors have a commonorigin because the multiple alpha-helical structures of the HLA bindingsite and the peptide it contains strongly resemble the triple helicalstructure of collagen. The natural killer receptors are postulated towork by a dimerisation mechanism with two receptors recognising twoseparate HLA sites on the cell which the natural killer cell isinvestigating. Possibly this dimerisation is part of the activation ordeactivation mechanism, depending on the class of receptor. In the caseof GPVI there may as well be the possibility for two GPVI molecules toassociate with one Fcγ, since each monomer of the Fey dimer has arecognition sequence. However, the stoichiometry is not yet known, andbased upon the structure of collagens, collagen-like peptides that actvia GPVI and convulxin, it seems likely that the strength of the signalis related to the number of GPVI/Fcγ complexes that are clusteredtogether. Other platelet receptors belonging to this Ig family includeICAM-2 (CD102) and PECAM (CD31).

All microorganisms, cell lines, expression systems, expression hosts,plasmids, promoters, resistance markers, replication origins,restriction sites or other fragments or parts of vectors which arementioned in the description not directly in connection with the claimedinvention are commercially or otherwise generally available. Providedthat no other hints are given, they are used only as examples and arenot essential with respect to the invention, and can be replaced byother to suitable tools and biological materials, respectively.

The techniques which are essential according to the invention aredescribed in detail below and above. Other techniques which are notdescribed in detail correspond to known standard methods which are wellknown to a person skilled is in the art, or are described more in detailin the cited references and patent applications and in the standardliterature (e.g. Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, 2nd Edition, Cold Spring Harbor; Harlow, Lane, 1988, Antibodies:A Laboratory Manual, Cold Spring Harbor).

EXAMPLES Example 1 Materials

Protein A-Sepharose, peroxidase-conjugated goat anti-mouse andanti-rabbit antibodies, bovine serum albumin, Crotalus durissusterrificus venom, wheat germ aggulutinin (WGA), N-hydroxysuccinimidylchloroformate-activated cross-linked 4% beaded agarose and Triton X-114were from Sigma Chemical Co. (St Louis, Mo.),Octanoyl-N-methyl-glucamide (ONMG) and nonanoyl-N-methyl-glucamide(NNMG) were from Oxyl Chemie (Bobingen, Germany).

Example 2 GPVI Isolation from Platelets

Membrane glycoproteins were isolated from platelets as previouslydescribed. Briefly, platelets (from 40 buffy coats) were washed andlysed in 2% Triton X-114 in the presence of protease inhibitors. TheTriton X-114 and aqueous phases were separated and the detergent phasewas loaded on a column of wheat-germ agglutinin coupled to Sepharose 4B.The platelet glycoproteins were eluted with 10 mM Tris HCl, pH 7.4, 30mM NaCl, 0.2% octanoyl-N-methylglucamide (ONMG) and 2%N-acetylglucosamine. After dialysis and concentration, the solution ofglycoproteins was loaded on a column of convulxin bound toN-hydroxylsuccinamidyl-p-nitrophenyl chloroformate activatedcross-linked 4% beaded agarose (1 mg/ml). The column was washed with 4volumes of 10 mM Tris HCl, pH7.4, 30 mM NaCl, 0.2%nonanoyl-N-methylglucamide (NNMG), and then with 4 volumes of 10 mM TrisHCl, pH7.4, 30 mM NaCl and 2% NNMG. GPVI was eluted with 0.08% SDS in 10mM Tris/HCl, pH 7.4. The solution was concentrated and loaded on apreparative gel of 8.5% polyacrylamide using the Model 491 Prep Cell(BioRad, CA). The preparative electrophoresis was performed undernon-reduced conditions following the manufacturer instructions. GPVIeluted as a single band at 65 kDa. The fractions were pooled,concentrated on Centricon-30 (Amicon, Beverly, Mass.) and resuspended in10 mM Tris/HCl, pH7.4 and 0.1% ONMG.

Example 3 Amino Acid Analysis of GPVI

GPVI was digested with the endoproteinases LysC and AspN (BoehringerMannheim, Germany). The 10 peptides generated were separated byreverse-phase HPLC and sequenced on an Applied Biosystem model 477Apulsed-liquid-phase protein sequencer with a model 120A on-linephenylthiohydantoin amino acid analyser.

Example 4 Amplification of a 221 bp Fragment Coding for Part of GPVIfrom a λgt11 cDNA Library

A sample (10¹⁰ pfu) (plaque forming units) from a human bone marrowlibrary (Clonetech, Palo Alto, Calif.) was amplified using 2combinations of 4 degenerate primers. The final primer concentrationswere 2 μM, the dNTP concentration was 200 μM and 2 U/100 μl reactionAmpliTaq Gold (Perkin Elmer, Rotkreuz, Switzerland) were used. The PCRconditions were 5 cycles at 37° C. followed by 30 cycles at 44° C. Thesense 19mer 5′ TYATHCCNGCNATGAARMG 3′ (SEQ ID NO: 4) and the antisense20mer 5′ TTRTANARNGCRAAYTGRTC 3′ (SEQ ID NO: 6) amplified a 221 bpfragment which was subcloned in Bluescript KS⁺ (Stratagene, La Jolla,Calif.) and sequenced using the T7 Sequenase kit (Amersham,Switzerland).

Example 5 Screening the Agt11 cDNA Library with the 221 bp GPVI Probe

The 221 bp fragment was cut from the plasmid, cleaned and labelled withα³²P-ATP (20 MBq/50 μl, Hartmann Analytik, Braunschweig, Germany) usingthe High Prime Labelling kit (Boehringer Mannheim, Switzerland). Thehuman bone marrow library was screened following the manufacturerinstructions. Positive phages were grown, their DNA isolated andsubcloned In BlueScript using either EcoRI or Sal 1 sites and sequenced.Sequencing was performed using the ABS system of RACE- Platelet poly ARNA was prepared as previously described (Power et al., Cytokine 7,479-482, 1995). Reverse transcription (30 μl) was performed using 5 μgof poly A RNA with the primer 5′TGAATGAGACGGTCAGTTCAGC 3′ (SEQ ID NO: 8)(20 μM), dNTP (1 mM), RNAsin (40 U), 1×AMV buffer and 20 U AMV reversetranscriptase for 20 min at 45° C. followed by 20 min at 52° C. Thereaction mixture was treated with 2 μl 6N NaOH at 65° C. for 30 min,neutralised with 2 μl 6N acetic acid, and concentrated in a Centricon 30(Amicon). An anchor was ligated to the first strand DNA following theprotocol of Aptes and Siebert (BioTechniques 15: 890-893, 1993). NestedPCR was performed using a primer complementary to the anchor and theprimer 5′ TTGTACAGAGCAAATTGGTC 3′(SEQ ID NO: 9) (35 cycles, 55° C.) andfollowed by the primer 5′ GACCAGAGGCTTCCGTTCTG 3′ (SEQ ID NO: 10) (30cycles at 53° C.). The highest band (350 bp) was separated by agaroseelectrophoresis from the lower ones, subcloned into BlueScript, andsequenced.

Example 6 Preparation of Anti-GPVI Fab and F(ab′)₂

Polyclonal antisera against human GPVI were generated in rabbits. IgGfrom rabbit anti-GPVI antiserum was purified as described. Digestion ofIgG with immobilized papain (Pierce) to generate Fab fragments wasperformed according to the standard protocol of the supplier. Fabfragments were separated from undigested IgG and Fc fragments using animmobilized Protein A (Sigma) column. The flowthrough was transferred toa dialysis tube, concentrated using solid polyethyleneglycol 20,000,thoroughly dialysed against 20 mM Hepes, 140 mM NaCl, 4 mM KCl, pH 7.4and stored at 4° C. until used. F(ab′)₂ fragments were prepared bypepsin digestion of IgG, 1:50 enzyme to substrate ratio (w/w), in 0.5 Macetate buffer, pH 4.0, at 37° C. for 18 hr. The pH was corrected to 7.4with diluted NaOH and the sample was dialysed against 20 mM phosphate,pH 7.4. F(ab′)₂ fragments were separated from undigested IgG and Fcfragments using Protein A chromatography. The flow-through wastransferred to dialysis tube, concentrated using solidpolyethyleneglycol 20 000, intensively dialysed against 20 mM Hepes, 140mM NaCl, 4 mM KCl, pH 7.4 and stored in aliquots at −20° C. Washedplatelets were lysed in Triton X-114 and phase separation was performedon the soluble material before isolating the membrane glycoproteinsassociated with the Triton X-114 phase by affinity chromatography onwheat germ agglutinin-Sepharose 4B as described previously. As GPVIrepresents a very small fraction of the platelet membrane glycoproteinpool, we used the specificity of the snake C-type lectin convulxin forisolation of this receptor. Affinity chromatography on convulxin coupledto Sepharose 4B yielded a 65 kDa protein as major product. However,uncharacterized material of both higher and lower Mr co-eluted with GPVIand could not be removed by extensive washing of the column. Preparativegel electrophoresis on 8.5% polyacrylamide was added as a final step ofpurification. Fractions containing GPVI were pooled and gave a singleband on reanalysis. Purified GPVI was tested for its ability to blockplatelet aggregation by collagen. A slight inhibitory effect wasobserved when aliquots of GPVI solution were added to the plateletsuspension. However, by preincubating GPVI with collagen before addingthe mixture to the platelet suspension, aggregation could be inhibitedin a dose-dependant manner. These platelets still aggregated when freshcollagen was added. Under non-reducing conditions, the isolated proteinhas a Mr of 62 kDa with a shift toward a slightly higher Mr (65 kDa)under reducing conditions. As the amino terminus of GPVI was found to beblocked, the protein was digested with the enzymes LysC and LysC/AspNwhich produced 4 and 6 peptides, respectively, from which sequence wasobtained. The peptides were separated by reverse phase HPLC on a C4column and sequenced using the Edman method. The amino acid sequences ofthese peptides are underlined in the translated cDNA sequence (FIG. 1).

1. An isolated DNA coding for Glycoprotein VI which comprises thesequence set forth in SEQ ID NO:
 1. 2. An isolated DNA which consists ofthe sequence set forth in SEQ ID NO: 1.